Project Summary/ Abstract Clonal hematopoiesis of indeterminate potential (CHIP) is defined as an expansion of somatic hematopoietic blood cell clone in individual without hematological disorders. Recent exome sequencing identified hematopoietic stem and progenitor cells (HSPCs) with frequent mutations of epigenetic regulators (e.g., the DNA methylcytosine dioxygenase TET2) that exhibited growth advantage with clonal expansion during aging. Interestingly, CHIP individuals with somatic TET2 mutations tend to have high risk of coronary cardiovascular diseases (CVD). This discovery heralds the advent of a molecular era in the dissection of novel pathogenic mechanisms underlying CHIP-CVD convergence. In animal studies that mimic clonal hematopoiesis, Tet2 LOF has been found to accelerate atherosclerosis and heart failure. While these studies provided detailed phenotypic characterizations, the underlying molecular mechanisms and the causal relations between TET2 LOF in CHIP and increased CVD risk remain largely unresolved. The PI?s laboratory has developed a set of unique tools to address this critical clinically-relevant knowledge gap, including (i) tissue specific Tet2-deficient mouse models (specific ablation of Tet2 in the myeloid lineage or in HSPCs) with reporter genes to enable real-time lineage tracing in vivo during cardiac injury; and (ii) dCas9 based epigenome editing tools that allow the interrogation of causal effects between epigenotypes and phenotypes. The team proposes to test the hypothesis that Tet2 controls the activity of enhancers that regulate the expression of key genes required for maintaining the proper function of monocytes/ macrophages in the reparative response to ischemic injury (e.g., myocardial infarction or MI). Aim 1 will address how Tet2 loss impairs myeloid cells and HSPCs that actively participate in the post-MI cardiac repair process. Aim 2 will address how Tet2 deficiency disrupts enhancer activities in key genes that are essential for proinflammatory to reparative monocyte conversion, thereby perturbing the biphasic post-MI response of monocyte to compromise timely resolution of inflammation and cardiac repair. The idea of restoring Tet2/5hmC function will be further tested to intervene post-MI tissue repair. This study introduces a new dimension to dissect CVD pathogenesis by focusing on the interplay between the cardiovascular system and the immune-hematopoietic system. Completion of this project is anticipated to yield novel insights on how somatic TET2 mutations-associated clonal hematopoiesis increases the risk of cardiovascular disease (CVD) and impairs cardiac function under stress. More clinically relevant, discoveries made in this study are also expected to establish the preclinical rationale for targeting defective epigenetic regulators to prevent and treat CVD.